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Abstract

Single-molecule fluorescence imaging has greatly contributed to our understanding of many bio-molecular systems. While reactions occurring in the range of several minutes can be readily studied using conventional single-molecule fluorescence microscopes, data acquisition for longer time scales is hindered by the focal drift of high numerical aperture objectives, which should be corrected in real time. Here, we developed a robust autofocusing system based on optical astigmatism analysis of single-molecule images. Compared to the previously developed methods, our approach has a merit of simplicity in that neither fiducial makers nor an additional laser-detector system is required. As a demonstration, we observed B-Z transition dynamics occurring for several hours.

Autofocusing from large defocuses. (a) Representative time traces of the fluorescence intensities (top; Cy3: green line, Cy5: red line), the position of the objective lens (middle), and the displacement of the objective lens for each step of autofocusing (bottom). The orange and black bars on top of the graphs indicate the on and off states of the autofocusing system, respectively. While the autofocusing system was off, the microscope was intentionally defocused downward or upward. (b) The autofocusing time for varying defocusing distances. Four different SNR conditions of Cy3 signal (2.0, 2.9, 4.3 and 6.1) were tested. The error bars were generated from five measurements. (c) The position stability of the objective lens while the image focus was maintained. Four different SNR conditions of Cy3 signal (2.0, 2.9, 4.3 and 6.1) were tested. The error bars indicate the standard error of the mean generated from 4 independent movies. The experiments were performed with a 0.1-s exposure time.

Focus maintenance. (a) Representative time traces of single molecule fluorescence intensities (top) and the SNR of the Cy3 signal (bottom). The experiment was performed with the autofocusing system turned off. (b) Representative time traces of the single-molecule fluorescence intensities (top panel), the SNR of the Cy3 signal (2nd panel), the FOM (3rd panel), the number of remaining Cy3 molecules (4th panel), and the position of the objective lens (bottom). The experiment was performed with the autofocusing system activated. The SNR was obtained by analyzing the nearest 50 frames of the Cy3 images (n = 10). The experiments were performed with a 3-s exposure time.

The real-time observation of B-Z transition kinetics at high-salt conditions. (a) The sequences of the DNA construct (B(CG)6B). (b) The experimental scheme. The interdye distance increases in the Z-form, resulting in decreased FRET. (c) Representative time traces of the fluorescence intensities (top) and the corresponding FRET (gray, bottom). The most probable FRET time trace generated via hidden Markov modeling (HMM) [23] is overlaid (blue, bottom). (d) FRET histograms showing Z-DNA formation at 4.0 M NaClO4. The histogram is fitted to two Gaussian functions. The low-FRET state (red) corresponds to Z-DNA, and the high-FRET state (green) corresponds to B-DNA. (e) The transition density plot at 4.0 M NaClO4 obtained from hidden Markov modeling [24]. (f) The salt dependence of the B-Z transition rates. The experiments were performed with a 3-s exposure time.